Methods and kits for determining ubiquitin protein ligase (E3) activity

Abstract

The present invention provides a method and an assay kit for detecting ubiquitin ligase (E3) activity in a sample. The method includes the steps of (1) preparing a sample mixture comprising ubiquitin, a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), a reaction buffer, and the sample; (2) incubating the sample mixture under conditions suitable for ubiquitination to occur; and (3) detecting a presence of ubiquitination products in the sample mixture after the incubation.

1 . A method for detecting ubiquitin ligase (E3) activity in a sample, said method comprising the steps of: 
 preparing a sample mixture comprising a ubiquitin, a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), a reaction buffer, and the sample;    preparing a control mixture comprising said E1, said E2, a ubiquitin ligase (E3), and said buffer;    incubating said sample mixture and said control mixture under conditions suitable for ubiquitination to occur; and    detecting a presence of ubiquitination product in said sample mixture and in said control mixture after the incubation.    
     
     
         2 . The method of  claim 1 , wherein said E2 is UbCH5b.  
     
     
         3 . The method of  claim 1 , wherein said mixture comprises a panel of E2s.  
     
     
         4 . The method of  claim 3 , wherein said panel of E2s comprises UbCH5b.  
     
     
         5 . The method of  claim 1 , wherein said ubiquitin is untagged ubiquitin.  
     
     
         6 . The method of  claim 5 , wherein the presence of ubiquitination product is detected using an anti-ubiquitin antibody.  
     
     
         7 . The method of  claim 1 , wherein said ubiquitin is tagged ubiquitin.  
     
     
         8 . The method of  claim 7 , wherein said ubiquitin is tagged with a His-tag.  
     
     
         9 . The method of  claim 8 , wherein the presence of ubiquitination product is detected using nickel-HRP.  
     
     
         10 . The method of  claim 1 , wherein said mixture further comprises a ubiquitination substrate.  
     
     
         11 . The method of  claim 1 , wherein said ubiquitin activating enzyme comprises a bacteria extract having E1 activity, wherein said E2 comprises a bacteria extract having UbCH5b activity, wherein said ubiquitin is tagged with a His-tag, and wherein the presence of ubiquitination product is detected using SDS-PAGE followed by blotting and detection with nickel-HRP.  
     
     
         12 . The method of  claim 1 , further comprising: 
 quantifying the ubiquitination product in said sample mixture and in said control mixture after incubation; and    determining E3 activity in said sample.    
     
     
         13 . An assay kit for detecting ubiquitin ligase (E3) activity, said kit comprising: 
 a ubiquitin activating enzyme (E1);    a ubiquitin conjugating enzyme (E2);    a ubiquitin;    a control ubiquitin ligase (E3);    a reaction buffer; and    an instruction for assay procedure.    
     
     
         14 . The kit of  claim 13 , wherein said E1 is a bacteria lysate having E1 activity, wherein said E2 is a bacteria lysate having UbCH5b activity, wherein said tagged ubiquitin or untagged ubiquitin is a bacteria lysate comprising ubiquitin.  
     
     
         15 . The assay kit of  claim 14 , wherein said ubiquitin comprises a His-tag.  
     
     
         16 . The assay kit of  claim 15 , further comprising nickel-HRP.  
     
     
         17 . The assay kit of  claim 14 , wherein said ubiquitin is one of untagged ubiquitin or tagged ubiquitin.  
     
     
         18 . The assay kit of  claim 17 , further comprising at least one detection agent selected from the group consisting of anti-ubiquitin antibodies, anti-Flag antibodies, anti-myc antibodies, anti-T7 antibodies, anti-HA antibodies, nickel-HRP, and ECL substrates.  
     
     
         19 . The assay kit of  claim 13 , further comprising a sample lysis buffer.  
     
     
         20 . The assay kit of  claim 13 , wherein said reaction buffer comprise Mg ++  and ATP